title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.
abstract: BACKGROUND
  Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication.
  
  
  METHODS
  To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference.
  
  
  RESULTS
  In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication.
  
  
  CONCLUSIONS
  Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication.
authors.affiliation: Laboratório de Pesquisas em Virologia, Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do Rio Preto-FAMERP, Av. Brigadeiro Faria Lima 5416, São José do Rio Preto, SP 15090-000, Brazil.
authors.forename: Ana Ts
authors.initials: ATs
authors.lastname: Morais
authors.forename: Ana Cb
authors.initials: AC
authors.lastname: Terzian
authors.forename: Danilo Vb
authors.initials: DV
authors.lastname: Duarte
authors.forename: Roberta Vm
authors.initials: RV
authors.lastname: Bronzoni
authors.forename: Maria Cfs
authors.initials: MC
authors.lastname: Madrid
authors.forename: Arieli F
authors.initials: AF
authors.lastname: Gavioli
authors.forename: Laura Hvg
authors.initials: LH
authors.lastname: Gil
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Oliveira
authors.forename: Cleslei F
authors.initials: CF
authors.lastname: Zanelli
authors.forename: Sandro R
authors.initials: SR
authors.lastname: Valentini
authors.forename: Paula
authors.initials: P
authors.lastname: Rahal
authors.forename: Mauricio L
authors.initials: ML
authors.lastname: Nogueira
mesh_terms: D004252:DNA Mutational Analysis; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D015005:Yellow fever virus / Q000502:physiology*
chemical_list: C443114:EIF3L protein, human; D039621:Eukaryotic Initiation Factor-3; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins